The effect of alpha1-adrenergic stimulation on the inotropy of the myocardium is still controversial. A positive inotropic effect has been well documented and it is often preceded by a short lived decrease in contractility. Additionally a persistent negative inotropic effect of alpha, stimulation has also been described in multicellular cardiac preparations. The purpose of this study was to characterize the effect of alpha1 stimulation on the cytosolic Ca transient and on the contractile properties of single ventricular myocytes under condition leading either to an enhancement or a decrease in twitch amplitude. Isolated rat ventricular myocytes, pretreated with propranolol were used to investigate the effect of alpha1-adrenergic stimulation with phenylephrine on contractility, cytosolic Ca homeostasis and on the frequency of spontaneous contractile waves (CW), which represent the mechanical expression of spontaneous calcium release from the sarcoplasmic reticulum (SR). a) In 1 mM bathing [Ca2=] (Ca), during field stimulation at 0.2 Hz, alpha had a positive inotropic effect which was reversibly abolished by prazosin and appeared related to an increased myofilament responsiveness to calcium in addition to some enhancement in the amplitude of the cytosolic Ca transient associated with the twitch. b) During stimulation at 0.2 Hz, in 5 mM Cao, CW appeared in some of the diastolic intervals. Phenylephrine abolished them and decreased twitch amplitude. In similar experiments with myocytes loaded with the Ca probe Indo-1 AM the negative effect of alpha1 on TA was associated with a decrease in the amplitude of the cytosolic Ca transient. c) In the absence of stimulation, in 5 mM Cao, alpha1 significantly and reversibly reduced cytosolic and CW frequency. This effect could be prevented by the alpha1 blocker prazosin. No effect of phenylephrine on cytosolic Ca was observed at rest in 1 mM Cao. These findings are consistent with the view that the effect of alpha1-adrenergic stimulation of cardiac cells can vary in relation to intracellular Ca.